Human erythropoietin (Ep) is an acidic glycoprotein hormone with an apparent molecular weight of 34,000 daltons. It is the primary regulator of erythrocyte (red blood cell) production. Its known major functions are promotion of erythroid differentiation and initiation of hemoglobin synthesis, but it may also be involved in stimulation of limited proliferation of immature erythrocyte precursors.
An understanding of the mode of Ep action is of considerable biological importance. Not only would it serve as a useful model for studying the differentiation and development of mammalian cells, but it would also be of great value in the diagnosis and treatment of anemias. Although much research has been directed to this area, progress has been slow due in part to the lack of pure Ep. This is caused both by scant availability of starting materials and by difficulties in purification.
Unavailability of sufficient quantities of pure Ep has also hindered the development of Ep-specific monoclonal antibodies using hybridoma techniques and the use of recombinant DNA technology in the molecular cloning of Ep genes and the production of hybrid cells which would produce human Ep gene products.
Ep circulates in the plasma space and is excreted in the urine at very low concentrations under normal conditions. However, under anemic or anoxic stress, Ep levels in the urine may increase considerably. Thus, urine from severely anemic patients (e.g., patients with aplastic anemia, leukemia, or various hemoglobinopathies) have been the sole source of human Ep, to date. Not all anemic patients, however, exhibit increased urinary Ep levels. Accordingly, monitoring of patients is necessary to determine whether their urine will be useful as a source of Ep. Moreover, once a patient responds to a therapeutic treatment, his or her urinary Ep levels change rapidly, making it necessary to seek a new Ep source. In addition, Ep must be purified from the urine before it can be further used.
Many attempts have been made in various laboratories to purify human Ep. The major difficulties with these attempts have been the limited supply of starting material, and the incomplete resolution of Ep from urinary contaminants. Early attempts to fractionate with organic solvents and salts resulted in a distribution of activity in several fractions. The fractions of higher activity have often been obtained in low yield. Conventional chromatographic techniques have been similarly limited in efficiency. Several purification procedures have been reported. One such procedure described by Espada, I., et al, Purification de Erythropoietina Urinaria Humana, Acta Physiol., Lat. Am. 10:122-129, 1970, involved a ten-step operation; briefly: (1) benzoic acid adsorption, (2) protein precipitation, (3) ethanol precipitation, (4) heat treatment, (5) Diethylaminoethyl(DEAE)-cellulose chromatography, (6) hydroxylapatite adsorption, (7) 2nd DEAE-cellulose chromatography, (8), (9), and (10) 1st, 2nd, and 3rd Sephadex G100 gel filtration. This procedure gave a 323-fold purification with 18.5% yield. The specific activity increased from 25 units/mg of protein in the starting material to 8086 units/mg of protein in the final product (units as defined below). According to these workers, this procedure is efficient only when applied to large amounts of raw material and when the starting material has an Ep titer of 20 units/mg or higher. The starting material used in the above-described work was urine collected in Argentina from patients afflicted with anemia due to hookworm infection.
Another procedure reported by Miyake, T, et al, Purification of Human Erythropoietin, J. Biol. Chem. 252:5558-5564, 1977, consisted of initial desalting on Sephadex G25, followed by seven steps, namely: (1) DEAE batch elution, (2) p-aminosalicylate treatment and phenol extraction, (3) ethanol fractionation, (4) DEAE agarose column chromatography, (5) sulfopropyl-Sephadex chromatography, (6) Sephadex G100 gel filtration, and (7) hydroxylapatite adsorption. Again, this procedure requires large amounts of starting sample with high initial specific activity. Seven million units with an exceptionally high starting Ep titer of 91 units/mg of protein were processed all at once. The final product had a specific activity of 70,400 units/mg of protein. This represented a purification factor of 930 with 21% yield.
High Ep titer urine from aplastic anemic patients of unknown origin collected in Kumanoto City, Japan was used as the starting material. In the United States, however, it is impossible to have a large supply of urine of such high Ep titer due to the practice of giving anemic patients blood transfusions. Thus, it is impossible to repeat this procedure on comparable starting material with equivalent Ep titer. Quite different results and much lower specific activity have been obtained when repeating this process on a small scale with low Ep titer urine samples collected in the U.S.A.
Furthermore, each of the above procedures requires constant use of large amounts of benzoic acid and phenol. The former is toxic, and the latter a known mutagen; they are thus deleterious to Ep research goals.
Aside from the problems due to the extremely low initial content of Ep in urine, purification of the hormone is quite difficult to achieve because it is contaminated with many urinary impurities with similar physiochemical properties. Many of the existing purification procedures are based on either conventional charge and size separations, or sugar-specific affinity to lectin derivatives. A simple prior group separation on the basis of a different and independent property, hydrophobicity, proved important for the elimination of contaminating impurities from Ep with similar size and charge as well as similar monosaccharide content. Use of hydrophobic interaction chromatography (HIC) in Ep purification, has been reported by Lee-Huang, S. A New Preparative Method for Isolation of Human Erythropoietin with Hydrophobic Interaction Chromatography, Blood 56: 620-624, 1980.
Immunoaffinity chromatography is highly specific and effective for the purification of many macromolecules. However, in the absence of sufficient quantities of pure Ep as the immunogen for the production and/or purification of Ep-specific antibodies, the potential of conventional immunoaffinity becomes limited. Even if highly purified Ep is used for the immunization of antibody producing animals, these animals frequently generate large amounts of antibodies against minor contaminants, especially when the main antigen is a weak immunogen, as is the case with Ep. Conventional immunoaffinity chromatography can thus only yield a preparation as pure as the original antigen, since antibodies to the contaminants also immunoabsorb their antigens.
The present invention involves a novel and simple immunoaffinity technique for use in Ep purification. The experimental results showed excellent potential and general applicability of the procedure. This novel procedure is especially well suited for initial processing of crude starting material of moderate Ep titer. In bypassing many steps, unnecessary handling of the sample is eliminated, and the yield is increased accordingly. The present specification includes a description of a systematic investigation of some of the important parameters for high resolution and good recovery. By the combination of HIC, Direct Immunoaffinity chromatography (DIAC), and Reverse Immunoaffinity Chromatography (RIAC) in particular, a purification factor of 35,000 fold with 59% yield has been achieved. The specific activity increased from 0.91 units/mg of protein in the starting material to 32,000 units/mg of protein in the final product. This procedure is simple, rapid, and effective, and is suitable for the processing of low and high Ep titered urine in large or small quantities. Some of the starting material was supplied by the National Heart, Lung, and Blood Institute. Additional urine samples were collected from various hospitals in New York City from patients suffering from disorders including aplastic anemia, hemolytic anemia, leukemia and various hemoglobinopathies.